Residue depletion of enrofloxacin and flumequine in feathers of broilers based on quantitative UHPLC-MS/MS detection.

To explore potential factors contributing to high fluoroquinolone resistance levels, it is essential to develop analytical methods capable of detecting residues and trace amounts of antibiotic use in broilers. The aim of the present study was to develop and in-house validate a sensitive UHPLC-MS/MS method capable of determining enrofloxacin (ENR) and flumequine (FLU) residues at slaughter age (day 45) when the animals were treated with these antimicrobials one day after hatching. Residue depletion of ENR and FLU in feathers was also assessed. Two experimental trials were performed, both consisting of 5 different treatment groups. In the first trial animals were treated with ENR and in the second one with FLU. The developed method was successfully validated and was found to be sensitive enough to detect residues of fluoroquinolones in the feathers up until slaughter age in all treatment groups. Average ENR concentration on day 45 was 10 ng g-1 feather after drinking water treatment, with all concentrations above the limit of quantification (LOQ) of 5 ng g-1 feather. For FLU average concentration on day 45 after drinking water administration was 4 ng g-1 feather, with an LOQ of 1 ng g-1 feather. Therefore, the method is suited for application to monitor fluoroquinolone use in broilers.

An Algoclay-Based Decontaminant Decreases Exposure to Aflatoxin B1, Ochratoxin A, and Deoxynivalenol in a Toxicokinetic Model, as well as Supports Intestinal Morphology, and Decreases Liver Oxidative Stress in Broiler Chickens Fed a Diet Naturally Contaminated with Deoxynivalenol.

The aims of this study were (i) to determine the effect of an algoclay-based decontaminant on the oral availability of three mycotoxins (deoxynivalenol; DON, ochratoxin A; OTA, and aflatoxin B1; AFB1) using an oral bolus model and (ii) to determine the effect of this decontaminant on the performance, intestinal morphology, liver oxidative stress, and metabolism, in broiler chickens fed a diet naturally contaminated with DON. In experiment 1, sixteen 27-day-old male chickens (approximately 1.6 kg body weight; BW) were fasted for 12 h and then given a bolus containing either the mycotoxins (0.5 mg DON/kg BW, 0.25 mg OTA/kg BW, and 2.0 mg AFB1/kg BW) alone (n = 8) or combined with the decontaminant (2.5 g decontaminant/kg feed; circa 240 mg/kg BW) (n = 8). Blood samples were taken between 0 h (before bolus administration) and 24 h post-administration for DON-3-sulphate, OTA, and AFB1 quantification in plasma. The algoclay decontaminant decreased the relative oral bioavailability of DON (39.9%), OTA (44.3%), and AFB1 (64.1%). In experiment 2, one-day-old male Ross broilers (n = 600) were divided into three treatments with ten replicates. Each replicate was a pen with 20 birds. The broiler chickens were fed a control diet with negligible levels of DON (0.19-0.25 mg/kg) or diets naturally contaminated with moderate levels of DON (2.60-2.91 mg/kg), either supplemented or not with an algoclay-based decontaminant (2 g/kg diet). Jejunum villus damage was observed on day 28, followed by villus shortening on d37 in broiler chickens fed the DON-contaminated diet. This negative effect was not observed when the DON-contaminated diet was supplemented with the algoclay-based decontaminant. On d37, the mRNA expression of glutathione synthetase was significantly increased in the liver of broiler chickens fed the DON-contaminated diet. However, its expression was similar to the control when the birds were fed the DON-contaminated diet supplemented with the algoclay-based decontaminant. In conclusion, the algoclay-based decontaminant reduced the systemic exposure of broiler chickens to DON, OTA, and AFB1 in a single oral bolus model. This can be attributed to the binding of the mycotoxins in the gastrointestinal tract. Moreover, dietary contamination with DON at levels between 2.69 and 2.91 mg/kg did not impair production performance but had a negative impact on broiler chicken intestinal morphology and the liver redox system. When the algoclay-based decontaminant was added to the diet, the harm caused by DON was no longer observed. This correlates with the results obtained in the toxicokinetic assay and can be attributed to a decreased absorption of DON.

Have We Neglected to Study Target-Site Drug Exposure in Children? A Systematic Review of the Literature.

BACKGROUND AND OBJECTIVE: Drug dosing should ideally be based on the drug concentrations at the target site, which, for most drugs, corresponds to the tissue. The exact influence of growth and development on drug tissue distribution is unclear. This systematic review compiles the current knowledge on the tissue distribution of systemically applied drugs in children, with the aim to identify priorities in tissue pharmacokinetic (PK) research in this population. METHODS: A systematic literature search was performed in the MEDLINE and Embase databases. RESULTS: Forty-two relevant articles were identified, of which 71% investigated antibiotics, while drug classes from the other studies were anticancer drugs, antifungals, anthelmintics, sedatives, thyreostatics, immunomodulators, antiarrhythmics, and exon skipping therapy. The majority of studies (83%) applied tissue biopsy as the sampling technique. Tonsil and/or adenoid tissue was most frequently examined (70% of all included patients). The majority of studies had a small sample size (median 9, range 1-93), did not include the youngest age categories (neonates and infants), and were of low reporting quality. Due to the heterogeneous data from different study compounds, dosing schedules, populations, and target tissues, the possibility for comparison of PK data between studies was limited. CONCLUSION: The influence of growth and development on drug tissue distribution continues to be a knowledge gap, due to the paucity of tissue PK data in children, especially in the younger age categories. Future research in this field should be encouraged as techniques to safely investigate drug tissue disposition in children are available.

Chestnut Wood Tannins in Broiler Diets: Pharmacokinetics, Serum Levels during Rearing, and Intestinal Absorption Pattern of Gallic Acid.

Studies on the bioavailability, serum levels, and absorption of hydrolyzable tannin compounds are lacking. In this study, we performed a pharmacokinetic trial, measured the serum levels of compounds in broilers that were reared with different feed added or not with tannins, and tested the digestibility of tannins throughout the intestinal tract. Only gallic acid and 4-O-methyl gallic acid were found in the serum. Moreover, gallic acid showed a 41.8% absolute oral bioavailability and a 72.3% relative bioavailability of gallic acid from chestnut extract compared to the standard. The rapid metabolization caused alternating serum levels during the day and night. These patterns were not affected by the feed type or the previous addition of tannins in the feed. The absorption and metabolization in the intestines occurred gradually throughout the intestinal tract. The latter was true for gallic acid as well as ellagic acid, which was not found in the serum. We can conclude that components from chestnut tannins are absorbed throughout all components of the intestinal tract and are eliminated quickly with little interaction from the feed and previous addition of tannins. Moreover, ellagic acid seems to be absorbed but would remain accumulated in the intestinal tissue or be metabolized by the microbiome.

In-vitro susceptibility and ex-vivo evaluation of macrocyclic lactone endectocides sub-lethal concentrations against Plasmodium vivax oocyst development in Anopheles arabiensis.

BACKGROUND: Asymptomatic malaria transmission has become a public health concern across malaria-endemic Africa including Ethiopia. Specifically, Plasmodium vivax is more efficient at transmitting earlier in the infection and at lower densities than Plasmodium falciparum. Consequently, a greater proportion of individuals infected with P. vivax can transmit without detectable gametocytaemia. Mass treatment of livestock with macrocyclic lactones (MLs), e.g., ivermectin and doramectin, was suggested as a complementary malaria vector tool because of their insecticidal effects. However, the effects of MLs on P. vivax in Anopheles arabiensis has not yet been fully explored. Hence, comparative in-vitro susceptibility and ex-vivo studies were conducted to evaluate the effects of ivermectin, doramectin and moxidectin sub-lethal concentrations on P. vivax oocyst development in An. arabiensis. METHODS: The 7-day sub-lethal concentrations of 25% (LC25) and 5% (LC5) were determined from in-vitro susceptibility tests on female An. arabiensis in Hemotek® membrane feeding assay. Next, an ex-vivo study was conducted using P. vivax gametocytes infected patient's blood spiked with the LC25 and LC5 of the MLs. At 7-days post-feeding, each mosquito was dissected under a dissection stereo microscope, stained with 0.5% (w/v) mercurochrome solution, and examined for the presence of P. vivax oocysts. Statistical analysis was based on a generalized mixed model with binomially distributed error terms. RESULTS: A 7-day lethal concentration of 25% (LC25, in ng/mL) of 7.1 (95% CI: [6.3;8.0]), 20.0 (95%CI:[17.8;22.5]) and 794.3 (95%CI:[716.4;1516.3]) were obtained for ivermectin, doramectin and moxidectin, respectively. Similarly, a lethal concentration of 5% (LC5, in ng/mL) of 0.6 (95% CI: [0.5;0.7]), 1.8 (95% CI:[1.6;2.0]) and 53.7 (95% CI:[ 48.4;102.5]) were obtained respectively for ivermectin, doramectin and moxidectin. The oocyst prevalence in treatment and control groups did not differ significantly (p > 0.05) from each other. Therefore, no direct effect of ML endectocides on P. vivax infection in An. arabiensis mosquitoes was observed at the sub-lethal concentration (LC25 and LC5). CONCLUSIONS: The effects of ivermectin and doramectin on malaria parasite is more likely via indirect effects, particularly by reducing the vectors lifespan and causing mortality before completing the parasite's sporogony cycle or reducing their vector capacity as it affects the locomotor activity of the mosquito.

Pharmacokinetics of gamma-hydroxybutyric acid in 6-week-old swine (Sus scrofa domesticus) after intravenous and oral administration.

Sedative as well as protective effects during hypoxia have been described for gamma-hydroxybutyric acid (GHB). Six swine (Sus scrofa domesticus) of 6 weeks old were administered NaGHB at a dose of 500 mg/kg intravenously (IV) and 500 and 750 mg/kg orally (PO) in a triple cross-over design. Repeated blood sampling was performed to allow pharmacokinetic analysis of GHB. Whole blood concentration at time point 0 after IV administration was 1727.21 ± 280.73 µg/mL, with a volume of distribution of 339.45 ± 51.41 mL/kg and clearance of 164.94 ± 47.05 mL/(kg h). The mean peak plasma concentrations after PO administration were 326.57 ± 36.70 and 488.01 ± 154.62 µg/mL for 500 mg/kg and 750 mg/kg, respectively. These were recorded at 1.42 ± 0.72 and 1.58 ± 0.58 h after PO dose for GHB 500 mg/kg and 750 mg/kg, respectively. The elimination half-life for IV and PO 500 mg/kg and PO 750 mg/kg dose was respectively 1.33 ± 0.30, 1.16 ± 0.31 and 1.11 ± 0.33 h. The bioavailability (F) for PO administration was 45%. No clinical adverse effects were observed after PO administration. Deep sleep was seen in one animal after IV administration, other animals showed head pressing and ataxia.

Pharmacokinetics of macrocyclic lactone endectocides in indigenous Zebu cattle and their insecticidal efficacy on Anopheles arabiensis.

Outdoor biting, outdoor resting, and early evening biting of Anopheles arabiensis is a challenge in current malaria control and elimination efforts in Africa. Zooprophylaxis using livestock treated with macrocyclic lactones is a novel approach to control zoophilic vectors. Therefore, the present study aimed to investigate the pharmacokinetics and insecticidal efficacy of ivermectin (IVER), doramectin (DORA), and moxidectin (MOXI) subcutaneous (SC) formulations in treated calves. The study was conducted using indigenous (Bos indicus) calves treated with SC formulation at a dosage of 0.5, 0.2 or 0.05 mg/kg body weight (BW) IVER or DORA and 0.2 or 0.05 mg/kg BW MOXI. Direct skin feeding of mosquitoes and animal blood sampling were performed at 4, 8, 12, and 24 h and on days 2, 3, 5, 7, 10, 14, 21, 28, and 35 post treatment. The survival of fully fed A. arabiensis mosquitoes was monitored for 10 days. Plasma samples were analyzed using UHPLC-MS/MS. A. arabiensis mortality percentages in the 0.5 mg/kg BW DORA and IVER groups were 65.74% (95% CI: [54.98; 76.50]) and 64.53% (95% CI: [53.77; 75.29]), respectively, over 35 days post treatment. At the recommended dose (0.2 mg/kg BW), promising overall A. arabiensis mortality rates of 61.79% (95% CI: [51.55; 72.03]) and 61.78% (95% CI: [51.02; 72.54]) were observed for IVER and DORA, respectively. In contrast, A. arabiensis mortality in the MOXI group was 50.23% (95% CI: [39.87, 60.58]). At 0.2 mg/kg BW dose, area under the plasma concentration versus time curve (AUC0-inf) values for IVER, DORA, and MOXI were 382.53 ± 133.25, 395.41 ± 132.12, and 215.85 ± 63.09 ng day/mL, respectively. An extended elimination half-life (T1/2el) was recorded for DORA (4.28 ± 0.93 d), at 0.2 mg/kg BW dose level, compared to that for IVER (3.16 ± 1.47 d). The T1/2el of MOXI was 2.17 ± 0.44 day. A maximum plasma concentration (Cmax) was recorded earlier for MOXI (10 h) than for IVER (1.6 days) and longer for DORA (3.0 days). For DORA and IVER, significant differences were found in T1/2el (P<0.05), Cmax (P<0.01), and AUC0-inf (P<0.01) between the higher 0.5 mg/kg BW and the lower 0.05 mg/kg BW doses. The T1/2el and AUC0-inf of DORA and IVER in the present study were significantly (p < 0.05) correlated with the observed insecticidal efficacy against A. arabiensis mosquitoes at 0.2 mg/kg a dose. Therefore, treating cattle with IVER or DORA could complement the malaria vector control interventions, especially in Ethiopia, where the zoophilic malaria vector A. arabiensis majorly contribute for residual malaria transmission.

Microdialysis as a safe and feasible method to study target-site piperacillin-tazobactam disposition in septic piglets and children.

OBJECTIVES: Knowledge on the tissue penetration of piperacillin-tazobactam in children with sepsis is lacking. In this study, the feasibility and performance of microdialysis experiments were explored in septic piglets and children as part of a translational research project. METHODS: Multiple-day microdialysis investigations were performed in muscle tissue of 22 piglets (of which 11 were septic) and 6 children with sepsis. An in vitro experiment preceded the (pre)clinical trials to derive optimal experimental settings and calibration technique. Linear mixed-effects models quantified the impact of sepsis on relative recovery (RR) and intercatheter, interindividual, interoccasion, and residual variability. RESULTS: In vivo microdialysis was well tolerated in piglets and children, with no significant adverse events reported. Using identical experimental settings, lower RR values were recorded in healthy and septic piglets (range: piperacillin, 17.2-29.1% and tazobactam, 23.5-29.1%) compared with the in vitro experiment (piperacillin, 43.3% and tazobactam, 55.3%), and there were unacceptably low values in children with sepsis (<10%). As a result, methodological changes were made in the pediatric trial. Realistic tissue concentration-time curves were derived in piglets and children. In piglets, sepsis reduced the RR. The greatest contributors to RR variability were residual (>40%) and interoccasion (>30%) variability. The internal standard method was the preferred calibration technique in both piglets and children. CONCLUSIONS: Microdialysis is a safe and applicable method for the measurement of tissue drug concentrations in piglets and children. This study demonstrated the impact of experimental settings, sepsis, and target population on individual RR.

Cefquinome shows a higher impact on the pig gut microbiome and resistome compared to ceftiofur.

Cephalosporins are licensed for treatment of severe bacterial infections in different species. However, the effect of these antimicrobials on the fecal microbiome and potential spread of resistance-associated genes causes great concern. This highlights the need to understand the impact of cephalosporins on the porcine fecal microbiome and resistome. A combination of long-read 16S rRNA gene and shotgun metagenomic sequencing was applied to investigate the effect of conventional treatment with either ceftiofur (3 mg.kg-1 intramuscular, 3 consecutive days) or cefquinome (2 mg.kg-1 intramuscular, 5 consecutive days) on the porcine microbiome and resistome. Fecal samples were collected from 17 pigs (6 ceftiofur treated, 6 cefquinome treated, 5 control pigs) at four different timepoints. Treatment with ceftiofur resulted in an increase in Proteobacteria members on microbiome level, while on resistome level selection in TetQ containing Bacteroides, CfxA6 containing Prevotella and blaTEM-1 containing Escherichia coli was observed. Cefquinome treatment resulted in a decline in overall species richness (α-diversity) and increase in Proteobacteria members. On genus level, administration of cefquinome significantly affected more genera than ceftiofur (18 vs 8). On resistome level, cefquinome resulted in a significant increase of six antimicrobial resistance genes, with no clear correlation with certain genera. For both antimicrobials, the resistome levels returned back to the control levels 21 days post-treatment. Overall, our study provides novel insights on the effect of specific cephalosporins on the porcine gut microbiome and resistome after conventional intramuscular treatment. These results might contribute to better tailoring of the most ideal treatment strategy for some bacterial infections.

Qualitative risk assessment of homogeneity, stability, and residual concentrations of antimicrobials in medicated feed and drinking water in pig rearing.

BACKGROUND: Despite the common use of oral group treatment in pig rearing, the magnitude of the factors influencing the homogeneity and stability of antimicrobial drugs in medicated feed and medicated drinking water are largely unknown, as well as the residual concentrations of the drugs after the end of the treatment. RESULTS: This study presents a qualitative risk assessment to estimate the magnitude of the risks for reduced homogeneity and stability, and increased residual concentrations of antimicrobial drugs in medicated feed and drinking water on the farm. Risk assessment was done using a questionnaire and farm visits (n = 52), combined with a second questionnaire, and concentrations of amoxicillin and doxycycline measured in medicated feed and water samples, each collected on 10 farms. For medicated feed, the duration of storage in the silo did not show to influence the concentration levels in a consistent trend, while the treatment duration had a low to negligible effect. A moderate to high risk was found caused by human error when preparing the medicated feed on the farm. Purchased medicated feed greatly reduces the risk of human error and drugs remain stable during the duration of treatment, while the risk of residual concentrations after the end of the treatment was estimated to be low to moderate. The feed intake variability was identified as a moderate to high risk factor. For medicated drinking water, the type of dosing pump, age of pre-solution, and human errors during the preparation of the pre-solution present a moderate to high risk on homogeneity and stability. Precipitation of the active substance in the absence of a stirrer in a drinking water tank was shown to be a low to moderate risk factor for residues after treatment. Waterline length had a weak correlation with the concentrations of the antimicrobials, while a moderate to high influence was detected for the water intake by the pigs. CONCLUSIONS: A considerable variation in drug concentration in both medicated feed and medicated drinking water was detected depending on their preparation. Therefore, it is important to know which factors influence the homogeneity and stability, and the residual concentrations after treatment.

Dose-dependent impact of enrofloxacin on broiler chicken gut resistome is mitigated by synbiotic application.

Fluoroquinolone agents are considered critical for human medicine by the World Health Organization (WHO). However, they are often used for the treatment of avian colibacillosis in poultry production, creating considerable concern regarding the potential spread of fluoroquinolone resistance genes from commensals to pathogens. Therefore, there is a need to understand the impact of fluoroquinolone application on the reservoir of ARGs in poultry gut and devise means to circumvent potential resistome expansion. Building upon a recent dose optimization effort, we used shotgun metagenomics to investigate the time-course change in the cecal microbiome and resistome of broiler chickens receiving an optimized dosage [12.5 mg/kg body weight (bw)/day], with or without synbiotic supplementation (PoultryStar®, BIOMIN GmbH), and a high dosage of enrofloxacin (50 mg/kg bw/day). Compared to the high dose treatment, the low (optimized) dose of enrofloxacin caused the most significant perturbations in the cecal microbiota and resistome of the broiler chickens, demonstrated by a lower cecal microbiota diversity while substantially increasing the antibiotic resistance genes (ARGs) resistome diversity. Withdrawal of antibiotics resulted in a pronounced reduction in ARG diversity. Chickens receiving the synbiotic treatment had the lowest diversity and number of enriched ARGs, suggesting an alleviating impact on the burden of the gut resistome. Some Proteobacteria were significantly increased in the cecal metagenome of chickens receiving enrofloxacin and showed a positive association with increased ARG burden. Differential abundance (DA) analysis revealed a significant increase in the abundance of ARGs encoding resistance to macrolides-lincosamides-streptogramins (MLS), aminoglycosides, and tetracyclines over the period of enrofloxacin application, with the optimized dosage application resulting in a twofold higher number of affected ARG compared to high dosage application. Our results provide novel insights into the dose-dependent effects of clinically important enrofloxacin application in shaping the broiler gut resistome, which was mitigated by a synbiotic application. The contribution to ameliorating the adverse effects of antimicrobial agents, that is, lowering the spread of antimicrobial resistance genes, on the poultry and potentially other livestock gastrointestinal microbiomes and resistomes merits further study.

Pharmacokinetic and pharmacodynamic properties of orally administered torasemide in healthy cats.

BACKGROUND: In people and dogs, torasemide has higher bioavailability, longer half-life, and longer duration of action than equivalent doses of furosemide but data regarding pharmacological properties of torasemide in cats are limited. OBJECTIVE: To assess pharmacokinetic and pharmacodynamic parameters of torasemide in healthy cats, and to investigate the effects of a single administration of torasemide on indicators of diuresis, plasma creatinine concentration, blood pressure, electrolyte concentrations and markers of the renin-angiotensin-aldosterone system (RAAS). ANIMALS: Six clinically healthy adult European shorthair cats. METHODS: Randomized 4-period crossover design with 3 groups and 4 treatments. Pharmacokinetic parameters were obtained using a noncompartmental analysis, and the clinically effective dose was assessed using a Hill model. RESULTS: Mean absolute bioavailability was estimated at 88.1%. Mean total body clearance was 3.64 mL/h/kg and mean terminal half-life was 12.9 hours. Urine output significantly increased after torasemide administration (P < .001). The urine sodium : potassium ratio (uNa : uK) paralleled and was statistically correlated to urine output (P < .001). Administration of a single torasemide dose led to a significant dose-dependent increase in urine aldosterone : creatinine ratio (uAldo : C; P < .001) and a transient decrease in plasma potassium concentration (P < .001) but did not affect blood pressure or plasma creatinine concentration. CONCLUSIONS AND CLINICAL IMPORTANCE: A single torasemide dose leads to a significant increase in diuresis and renin-angiotensin-aldosterone system (RAAS) activation in healthy cats, with high absolute bioavailability, and without clinically relevant adverse effects. Pharmacokinetic parameters indicate that once daily dosing of 0.27 mg/kg may be appropriate in a clinical setting.

Pharmacokinetics of Cannabidiol Following Intranasal, Intrarectal, and Oral Administration in Healthy Dogs.

The therapeutic potential of cannabidiol (CBD), a non-psychtropic component of the Cannabis sativa plant, is substantiated more and more. We aimed to determine the pharmacokinetic behavior of CBD after a single dose via intranasal (IN) and intrarectal (IR) administration in six healthy Beagle dogs age 3-8 years old, and compare to the oral administration route (PO). Standardized dosages applied for IN, IR and PO were 20, 100, and 100 mg, respectively. Each dog underwent the same protocol but received CBD through a different administration route. CBD plasma concentrations were determined by ultra-high performance liquid chromatography-tandem mass spectrometry before and at fixed time points after administration. Non-compartmental analysis was performed on the plasma concentration-time profiles. Plasma CBD concentrations after IR administration were below the limit of quantification. The mean area under the curve (AUC) after IN and PO CBD administration was 61 and 1,376 ng/mL*h, respectively. The maximal plasma CBD concentration (Cmax) after IN and PO CBD administration was 28 and 217 ng/mL reached after 0.5 and 3.5 h (Tmax), respectively. Significant differences between IN and PO administration were found in the Tmax (p = 0.04). Higher AUC and Cmax were achieved with 100 mg PO compared to 20 mg IN, but no significant differences were found when AUC (p = 0.09) and Cmax (p = 0.44) were normalized to 1 mg dosages. IN administration of CBD resulted in faster absorption when compared to PO administration. However, PO remains the most favorable route for CBD delivery due to its more feasible administration. The IR administration route is not advised for clinical application.

Intestinal Exposure to Ceftiofur and Cefquinome after Intramuscular Treatment and the Impact of Ceftiofur on the Pig Fecal Microbiome and Resistome.

Optimization of antimicrobial treatment during a bacterial infection in livestock requires in-depth knowledge of the impact of antimicrobial therapy on the pathogen and commensal microbiota. Once administered antimicrobials and/or their metabolites are excreted either by the kidneys through urine and/or by the intestinal tract through feces, causing antimicrobial pressure and possibly the emergence of resistance in the gastro-intestinal tract. So far, the excretion of ceftiofur and cefquinome in the intestinal tract of pigs has not been described. The objective of this study was to investigate the excretion of ceftiofur and cefquinome in the different segments of the gut and feces after intramuscular administration. Therefore, 16 pigs were treated either with ceftiofur (n = 8) or cefquinome (n = 8), and feces were collected during the entire treatment period. The presence of ceftiofur and desfuroylceftiofuracetamide or cefquinome were quantified via liquid chromatography−tandem mass spectrometry. At the end of the treatment, pigs were euthanized, and samples from the duodenum, jejunum, ileum and cecum were analyzed. In feces, no active antimicrobial residues could be measured, except for one ceftiofur-treated pig. In the gut segments, the concentration of both antimicrobials increased from duodenum toward the ileum, with a maximum in the ileum (187.8 ± 101.7 ng·g−1 ceftiofur-related residues, 57.8 ± 37.5 ng·g−1 cefquinome) and sharply decreased in the cecum (below the limit of quantification for ceftiofur-related residues, 6.4 ± 4.2 ng·g−1 cefquinome). Additionally, long-read Nanopore sequencing and targeted quantitative polymerase chain reaction (qPCR) were performed in an attempt to clarify the discrepancy in fecal excretion of ceftiofur-related residues between pigs. In general, there was an increase in Prevotella, Bacteroides and Faecalibacterium and a decrease in Escherichia and Clostridium after ceftiofur administration (q-value < 0.05). The sequencing and qPCR could not provide an explanation for the unexpected excretion of ceftiofur-related residues in one pig out of eight. Overall, this study provides valuable information on the gut excretion of parenteral administered ceftiofur and cefquinome.

Development and Validation of a Reliable UHPLC-MS/MS Method for Simultaneous Quantification of Macrocyclic Lactones in Bovine Plasma.

A fast, accurate and reliable ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed for simultaneous quantification of ivermectin (IVER), doramectin (DORA), and moxidectin (MOXI) in bovine plasma. A priority for sample preparation was the eradication of possible infectious diseases to avoid travel restrictions. The sample preparation was based on protein precipitation using 1% formic acid in acetonitrile, followed by Ostro® 96-well plate pass-through sample clean-up. The simple and straightforward procedure, along with the short analysis time, makes the current method unique and suitable for a large set of sample analyses per day for PK studies. Chromatographic separation was performed using an Acquity UPLC HSS-T3 column, with 0.01% acetic acid in water and methanol, on an Acquity H-Class ultra-high performance liquid chromatograph (UHPLC) system. The MS/MS instrument was a Xevo TQ-S® mass spectrometer, operating in the positive electrospray ionization mode and two multiple reaction monitoring (MRM) transitions were monitored per component. The MRM transitions of m/z 897.50 > 753.4 for IVER, m/z 921.70 > 777.40 for DORA and m/z 640.40 > 123.10 for MOXI were used for quantification. The method validation was performed using matrix-matched calibration curves in a concentration range of 1 to 500 ng/mL. Calibration curves fitted a quadratic regression model with 1/x2 weighting (r ≥ 0.998 and GoF ≤ 4.85%). Limits of quantification (LOQ) values of 1 ng/mL were obtained for all the analytes, while the limits of detection (LOD) were 0.02 ng/mL for IVER, 0.03 ng/mL for DORA, and 0.58 ng/mL for MOXI. The results of within-day (RSD < 6.50%) and between-day (RSD < 8.10%) precision and accuracies fell within acceptance ranges. No carry-over and no peak were detected in the UHPLC-MS/MS chromatogram of blank samples showing good specificity of the method. The applicability of the developed method was proved by an analysis of the field PK samples.

Population Pharmacokinetics of Intravenous Amoxicillin Combined With Clavulanic Acid in Healthy and Critically Ill Dogs.

Background: Data regarding antimicrobial pharmacokinetics (PK) in critically ill dogs are lacking and likely differ from those of healthy dogs. The aim of this work is to describe a population PK model for intravenous (IV) amoxicillin-clavulanic acid (AMC) in both healthy and sick dogs and to simulate a range of clinical dosing scenarios to compute PK/PD cutoffs for both populations. Methods: This study used a prospective clinical trial in normal and critically ill dogs. Twelve client-owned dogs hospitalized in the intensive care unit (ICU) received IV AMC 20 mg/kg every 8 h (0.5-h infusion) during at least 48 h. Eight blood samples were collected at predetermined times, including four trough samples before the next administration. Clinical covariates and outcome were recorded, including survival to discharge and bacteriologic clinical failure. Satellite PK data were obtained de novo from a group of 12 healthy research dogs that were dosed with a single AMC 20 mg/kg IV. Non-linear mixed-effects model was used to estimate the PK parameters (and the effect of health upon them) together with variability within and between subjects. Monte Carlo simulations were performed with seven dosage regimens (standard and increased doses). The correlation between model-derived drug exposure and clinical covariates was tested with Spearman's non-parametric correlation analysis. Outcome was recorded including survival to discharge and bacteriologic clinical failure. Results: A total of 218 amoxicillin concentrations in plasma were available for healthy and sick dogs. A tricompartmental model best described the data. Amoxicillin clearance was reduced by 56% in sick dogs (0.147 L/kg/h) compared with healthy dogs (0.336 L/kg/h); intercompartmental clearance was also decreased (p <0.01). None of the clinical data covariates were significantly correlated with individual exposure. Monte Carlo simulations showed that higher PK/PD cutoff values of 8 mg/L could be reached in sick dogs by extending the infusion to 3 h or doubling the dose. Conclusions: The PK of AMC is profoundly different in critically ill dogs compared with normal dogs, with much higher interindividual variability and a lower systemic clearance. Our study allows to generate hypotheses with regard to higher AMC exposure in clinical dogs and provides supporting data to revise current AMC clinical breakpoint for IV administration.

Development and Validation of Liquid Chromatography-Tandem Mass Spectrometry Methods for the Quantification of Cefquinome, Ceftiofur, and Desfuroylceftiofuracetamide in Porcine Feces with Emphasis on Analyte Stability.

Cefquinome and ceftiofur are ß-lactam antibiotics used for the treatment of bacterial infections in swine. Although these antimicrobials are administered intramuscularly, the exposure of the gut microbiota to these cephalosporins is not well described. This exposure can contribute to the emergence and spread of antimicrobials in the environment and to the possible spread of antimicrobial resistance genes. To assess the impact of drug administration on the intestinal excretion of these antimicrobials it is essential to measure the amounts of native compound and metabolites in feces. Two (ultra)-high-performance liquid chromatography-tandem mass spectrometry ((U)HPLC-MS/MS) methods were developed and validated, one for the determination of cefquinome and ceftiofur and the other for the determination of ceftiofur residues, measured as desfuroylceftiofuracetamide, in porcine feces. The matrix-based calibration curve was linear from 5 ng g-1 to 1000 ng g-1 for cefquinome (correlation coefficient (r) = 0.9990 ± 0.0007; goodness of fit (gof) = 3.70 ± 1.43) and ceftiofur (r = 0.9979 ± 0.0009; gof = 5.51 ± 1.14) and quadratic from 30 ng g-1 to 2000 ng g-1 for desfuroylceftiofuracetamide (r = 0.9960 ± 0.0020; gof = 7.31 ± 1.76). The within-day and between-day precision and accuracy fell within the specified ranges. Since ß-lactam antibiotics are known to be unstable in feces, additional experiments were conducted to adjust the sampling protocol in order to minimize the impact of the matrix constituents on the stability of the analytes. Immediately after sampling, 500 µL of an 8 µg mL-1 tazobactam solution in water was added to 0.5 g feces, to reduce the degradation in matrix.

Enrofloxacin Dose Optimization for the Treatment of Colibacillosis in Broiler Chickens Using a Drinking Behaviour Pharmacokinetic Model.

Enrofloxacin is frequently administered via drinking water for the treatment of colibacillosis in broiler chickens. However, the EMA/CVMP has urged to re-evaluate historically approved doses, especially for antimicrobials administered via drinking water. In response, the objectives of this study were two-fold. First, to evaluate the pharmacokinetics (PK) of enrofloxacin following IV, PO and drinking water administration. Second, to predict the efficacy of a range of doses in the drinking water for the treatment of APEC infections. For the first objective, PK parameters were estimated by fitting a one-compartmental model with a zero-order IV infusion and an oral absorption lag function to the simultaneously modelled IV and PO data. After fixing these parameter values, a drinking behaviour pharmacokinetic (DBPK) model was developed for the description and prediction of drinking water PK profiles by adding three model improvements (different diurnal and nocturnal drinking rates, inter-animal variability in water consumption and taking account of dose non-proportionality). The subsequent simulations and probability of target attainment (PTA) analysis predicted that a dose of 12.5 mg/kg/24 h is efficacious in treating colibacillosis with an MIC up to 0.125 µg/mL (ECOFF), whereas the currently registered dose (10 mg/kg/24 h) reaches a PTA of 66% at ECOFF.

The Development of a Juvenile Porcine Augmented Renal Clearance Model Through Continuous Infusion of Lipopolysaccharides: An Exploratory Study.

Augmented renal clearance (ARC) as observed in the critically ill (pediatric) population can have a major impact on the pharmacokinetics and posology of renally excreted drugs. Although sepsis has been described as a major trigger in the development of ARC in human critically ill patients, mechanistic insights on ARC are currently lacking. An appropriate ARC animal model could contribute to reveal these underlying mechanisms. In this exploratory study, a state of ARC was induced in 8-week-old piglets. Conscious piglets were continuously infused over 36 h with lipopolysaccharides (LPS) from Escherichia coli (O111:B4) to induce sepsis and subsequently trigger ARC. To study the dose-dependent effect of LPS on the renal function, three different doses (0.75, 2.0, 5.0 µg/kg/h) were administered (two ♂ piglets/dose, one sham piglet), in combination with fluid administration (0.9% NaCl) at 6 ml/kg/h. Single boluses of renal markers, i.e., creatinine [40 mg/kg body weight (BW)], iohexol (64.7 mg/kg BW), and para-aminohippuric acid (PAH, 10 mg/kg BW) were administered intravenously to evaluate the effect of LPS on the renal function. Clinical parameters were monitored periodically. Blood sampling was performed to determine the effect on hematology, neutrophil gelatinase-associated lipocalin, and prostaglandin E2 plasma levels. All piglets that were continuously infused with LPS displayed an elevated body temperature, heart rhythm, and respiratory rate ~1-3 h after start of the infusion. After infusion, considerably higher total body clearances of iohexol, creatinine, and PAH were observed, independent of the administration of LPS and/or its dose. Since also the sham piglet, receiving no LPS, demonstrated a comparable increase in renal function, the contribution of fluid administration to the development of ARC should be further evaluated.

Volumetric absorptive microsampling as alternative sampling technique for renal function assessment in the paediatric population using iohexol.

The glomerular filtration rate (GFR) is considered the best overall index for the renal function. Currently, one of the most promising exogenous markers for GFR assessment is iohexol. In this study, the suitability of volumetric absorptive microsampling (VAMS) as alternative for the conventional blood sampling and quantification of iohexol in paediatric plasma was assessed. Therefore, a new, fully validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed. Subsequently, the clinical suitability was evaluated in 20 paediatric patients by comparing plasma iohexol concentrations and associated GFR values obtained by the VAMS method with those obtained by conventional blood sampling and quantification of iohexol in plasma. The developed, simple and cost-effective LC-MS/MS-method fulfilled all pre-set validation acceptance criteria. Iohexol could be accurately quantified within a haematocrit range of 20-60% and long-term stability of iohexol in VAMS was demonstrated up to 245 days under different storage temperatures. Both iohexol plasma concentrations (r = 0.98, mean bias: -4.20%) and derived GFR values (r = 0.99; mean bias: 1.31%), obtained by a conventional plasma and the VAMS method, demonstrated good correlation and acceptable bias. The agreement between the two methods was especially good for GFR values higher than 60 mL/min/1.73 m2. Nevertheless, for GFR values <60 mL/min/1.73 m2 the accuracy compared to the plasma method was lower. However, small adjustments to the sampling protocol could probably solve this problem.